Approximately 1/3 of the population is affected by chronic periodontitis, a bacterially induced destruction of the attachment of the tooth to bone. The gingival crevice is host to a large number of bacteria, most of them currently uncultivated. The pathogenesis of periodontitis, although not well understood, is thought to involve polymicrobial consortia that appear to include many of these uncultivated bacteria. The gingival crevice offers colonizing bacteria a complex environment that includes a non-shedding tooth surface, a continuously desquamating epithelial wall covering the underlying connective tissue, and the narrow fluid-filled space in between. Many species of bacteria have been shown to attach to or invade the tissues, and these invasive bacteria are probably shed from the tooth-associated biofilm. Both the biofilm community and invasive bacteria are assumed to play an important role in pathogenesis. The goal of this project is to compare the bacterial community profiles found in periodontal health and disease from each of the physical compartments in the gingival crevice: the tooth, the crevicular fluid, and the tissue. Comparisons across disease status and physical location will elucidate the most important species and the most critical physical location for pathogens or beneficial species and further our understanding of the dynamics within this complex microbial community. A combination of rDNA 16S cloning and sequencing, PCR, and real-time PCR will be used for these studies. Experiments to further our understanding of polymicrobial interactions are also planned. Data on presence of cultivated and uncultivated species will be obtained by PCR, and analyzed for bacterial co- associations using a new statistical approach, Cluster Analysis for Categorical Data (CACD). This approach was developed by our research team to determine physically co-associated complexes of bacteria. These experiments should bring us closer to understanding the complex interactions that make up the crevicular ecosystem and biofilm. Finally, using the tools developed in the first two aims we will cultivate and characterize as-yet-uncultivated species that show associations with periodontitis or health. We will isolate these bacterial species using a combination of newly developed cultivation procedures and molecular tracking methods. The proposed studies will bring us closer to understanding the bacterial etiology of periodontitis, and closer to targeted therapies to promote health and treat disease.